Multiple Myeloma (MM) is a
heterogeneous disease with a strong need for robust markers for
prognosis. Frequently occurring chromosomal abnormalities, such as
t(4;14), gain(1q), and del(17p) etc. have some prognostic power,
but lack robustness across different cohorts. Alternatively, gene
expression profiling (GEP) studies have developed specific high
risk signatures such as the SKY92 (EMC92, Kuiper et al. Leukemia
2012), which has shown to be a robust prognostic factor across five
different clinical datasets. Moreover, studies comparing prognostic
markers have indicated that the SKY92 signature outperforms all
other markers for identifying high risk patients, both in single
and multivariate analyses. Similarly, when assessing the prognostic
value of combinations of various prognostic markers, the SKY92
combined with ISS was the top performer, and also enables detection
of a low risk group (Kuiper et al. ASH 2014). Here, we present a
further validation of the low and high risk groups identified by
the SKY92 signature in combination with ISS on two additional
cohorts of patients with diverse treatment backgrounds, containing
newly diagnosed, previously treated, and relapsed/refractory MM
patients.

Materials and
Methods

The SKY92 signature was applied to
two independent datasets. Firstly, the dataset from the Total
Therapy 6 (TT6) trial, which is a phase 2 trial for symptomatic MM
patients who have received 1 or more prior lines of treatment. The
TT6 treatment regime consists of VTD-PACE induction, double
transplant with Melphalan + VRD-PACE, followed by alternating
VRD/VMD maintenance. Affymetrix HG-U133 Plus 2.0 chips were
performed at baseline for n=55 patients, and OS was made available
previously (Gene Expression Omnibus identifier: GSE57317). However,
ISS was not available for this dataset. Secondly, a dataset of
patients enrolled at two hospitals in the Czech Republic, and one
in Slovakia (Kryukov et al. Leuk&Lymph 2013). Patients of all
ages, and from first line up to seventh line of treatment were
included (treatments incl Bort, Len, Dex). For n=73 patients
Affymetrix Human Gene ST 1.0 array, OS (n=66), and ISS (n=58) was
made available previously (ArrayExpress accession number:
E-MTAB-1038). Both datasets were processed from .CEL files by MAS5
(TT6), and RMA (Czech), followed by mean variance normalization per
probeset across the patients. The SKY92 was applied as previously
described (Kuiper et al. Leukemia 2012), and identifies a High Risk
and Standard Risk group. In conjunction with ISS, the SKY92
Standard Risk group is then further stratified into low and
intermediate risk groups (Kuiper et al. ASH 2014). Kaplan-Meier
plots were created, and the Cox proportional hazards model was used
to calculate Hazard Ratios (HR), and associated 1-sided p-values
that assess whether the SKY92 High Risk group has worse survival
than SKY92 Standard Risk group (i.e. HR>1).

Results

Figure 1 shows the Kaplan Meier
plots of the SKY92 High Risk and Standard Risk groups on the TT6
and Czech cohorts. On the TT6 dataset, the SKY92 signature
identifies 11 out of 55 patients (20%) as High Risk. In both
datasets, the SKY92 High Risk group has significantly worse overall
survival, HR=10.3, p=7.4 * 10-6 (TT6), and HR=2.6, p=2.2
* 10-2 (Czech). In addition, the combination of SKY92
with ISS on the Czech dataset identifies a low risk group of 14 out
of 61 patients (23%), with a five year overall survival estimate of
100% versus 28.7% in the SKY92 High Risk group (HR=inf). Robustness
of the SKY92 signature is further demonstrated by the fact that it
validates on both datasets, despite different microarray platforms
being used.

Conclusions

The SKY92 high risk signature has
been successfully validated on two independent datasets generated
using different microarray platforms. In addition, on the Czech
data, the low risk group (SKY92 Standard Risk combined with ISS 1)
has been successfully validated. Together, this signifies the
robust nature of the SKY92 signature for high and low risk
prediction, across treatments, and with applicability in newly
diagnosed, treated, and relapsed/refractory MM patients.

Figure 1. Kaplan-Meier plots
showing a significantly poorer overall survival in patients
identified as SKY92 High Risk (red curves), relative to SKY92
Standard Risk, on both the TT6 (left), and Czech (middle) datasets,
as well as a low risk group by SKY92 & ISS1 on the Czech
dataset (green curve, right).